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1.
Front Cell Infect Microbiol ; 11: 705468, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34490144

RESUMO

Enterotoxigenic Escherichia coli (ETEC) is a WHO priority pathogen and vaccine target which causes infections in low-income and middle-income countries, travelers visiting endemic regions. The global urgent demand for an effective preventive intervention has become more pressing as ETEC strains have become increasingly multiple antibiotic resistant. However, the vaccine development pipeline has been slow to address this urgent need. To date, vaccine development has focused mainly on canonical antigens such as colonization factors and expressed toxins but due to genomic plasticity of this enteric pathogen, it has proven difficult to develop effective vaccines. In this study, we investigated the highly conserved non-canonical vaccine candidate YghJ/SsLE. Using the mass spectrometry-based method BEMAP, we demonstrate that YghJ is hyperglycosylated in ETEC and identify 54 O-linked Set/Thr residues within the 1519 amino acid primary sequence. The glycosylation sites are evenly distributed throughout the sequence and do not appear to affect the folding of the overall protein structure. Although the glycosylation sites only constitute a minor subpopulation of the available epitopes, we observed a notable difference in the immunogenicity of the glycosylated YghJ and the non-glycosylated protein variant. We can demonstrate by ELISA that serum from patients enrolled in an ETEC H10407 controlled infection study are significantly more reactive with glycosylated YghJ compared to the non-glycosylated variant. This study provides an important link between O-linked glycosylation and the relative immunogenicity of bacterial proteins and further highlights the importance of this observation in considering ETEC proteins for inclusion in future broad coverage subunit vaccine candidates.


Assuntos
Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Metaloproteases , Antígenos de Bactérias , Epitopos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosilação , Humanos , Metaloproteases/genética , Metaloproteases/metabolismo
2.
J Enzyme Inhib Med Chem ; 36(1): 1829-1838, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34353202

RESUMO

As the largest organ in human body, skin acts as a physicochemical barrier, offering protection against harmful environmental stressors, such as chemicals, pathogens, temperature and radiation. Nonetheless, skins prominence goes further, with a significant psychosocial role in an increasingly ageing population. Prompted by consumers' concern regarding skincare, cosmetic industry has been developing new formulas capable of lessening the most visible signs of ageing, including reduction in skin density and elasticity, wrinkling and hyperpigmentation. Allied to skincare is the rising importance set on natural products, sustainably obtained from less environmental impacting methods. Cyanobacteria and microalgae are adding importance in this field, given their ability to biosynthesize secondary metabolites with anti-ageing potential. In this review, we present an overview on the potential of cyanobacteria and microalgae compounds to overcome skin-ageing, essentially by exploring their effects on the metalloproteinases collagenase, elastase, gelatinase and hyaluronidase, and in other enzymes involved in the pigmentation process.


Assuntos
Antioxidantes/química , Misturas Complexas/química , Cianobactérias/química , Matriz Extracelular/metabolismo , Hiperpigmentação/tratamento farmacológico , Microalgas/química , Envelhecimento da Pele/efeitos dos fármacos , Antioxidantes/farmacologia , Produtos Biológicos/química , Colagenases/metabolismo , Misturas Complexas/farmacologia , Gelatinases/metabolismo , Humanos , Hialuronoglucosaminidase/metabolismo , Metaloproteases/metabolismo , Elastase Pancreática/metabolismo , Pele
3.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445715

RESUMO

Breast cancer continues to be one of the main causes of morbidity and mortality globally and was the leading cause of cancer death in women in Spain in 2020. Early diagnosis is one of the most effective methods to lower the incidence and mortality rates of breast cancer. The human metalloproteinases (MMP) mainly function as proteolytic enzymes degrading the extracellular matrix and plays important roles in most steps of breast tumorigenesis. This retrospective cohort study shows the immunohistochemical expression levels of MMP-1, MMP-2, MMP-3, and MMP-9 in 154 women with breast cancer and 42 women without tumor disease. The samples of breast tissue are assessed using several tissue matrices (TMA). The percentages of staining (≤50%->50%) and intensity levels of staining (weak, moderate, or intense) are considered. The immunohistochemical expression of the MMP-1-intensity (p = 0.043) and MMP-3 percentage (p = 0.018) and intensity, (p = 0.025) present statistically significant associations with the variable group (control-case); therefore, expression in the tumor tissue samples of these MMPs may be related to the development of breast cancer. The relationships between these MMPs and some clinicopathological factors in breast cancer are also evaluated but no correlation is found. These results suggest the use of MMP-1 and MMP-3 as potential biomarkers of breast cancer diagnosis.


Assuntos
Neoplasias da Mama/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Mama/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Estudos de Casos e Controles , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica/métodos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Pessoa de Meia-Idade , Estudos Retrospectivos , Espanha , Inibidores Teciduais de Metaloproteinases/metabolismo
4.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209086

RESUMO

Mesenchymal stem cells (MSCs) obtained from various sources, including bone marrow, have been proposed as a therapeutic strategy for the improvement of tissue repair/regeneration, including the repair of cartilage defects or lesions. Often the highly inflammatory environment after injury or during diseases, however, greatly diminishes the therapeutic and reparative effectiveness of MSCs. Therefore, the identification of novel factors that can protect MSCs against an inflammatory environment may enhance the effectiveness of these cells in repairing tissues, such as articular cartilage. In this study, we investigated whether a peptide (P15-1) that binds to hyaluronan (HA), a major component of the extracellular matrix of cartilage, protects bone-marrow-derived MSCs (BMSCs) in an inflammatory environment. The results showed that P15-1 reduced the mRNA levels of catabolic and inflammatory markers in interleukin-1beta (IL-1ß)-treated human BMSCs. In addition, P15-1 enhanced the attachment of BMSCs to HA-coated tissue culture dishes and stimulated the chondrogenic differentiation of the multipotential murine C3H/10T1/2 MSC line in a micromass culture. In conclusion, our findings suggest that P15-1 may increase the capacity of BMSCs to repair cartilage via the protection of these cells in an inflammatory environment and the stimulation of their attachment to an HA-containing matrix and chondrogenic differentiation.


Assuntos
Anti-Inflamatórios/farmacologia , Proteínas da Matriz Extracelular/química , Receptores de Hialuronatos/química , Ácido Hialurônico/metabolismo , Interleucina-1beta/efeitos adversos , Células-Tronco Mesenquimais/citologia , Peptídeos/farmacologia , Animais , Anti-Inflamatórios/química , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Condrogênese , Ciclo-Oxigenase 2/genética , Regulação da Expressão Gênica , Humanos , Interleucina-6/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Metaloproteases/genética , Camundongos , Peptídeos/química
5.
Toxins (Basel) ; 13(5)2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066626

RESUMO

The venoms of small rear-fanged snakes (RFS) remain largely unexplored, despite increased recognition of their importance in understanding venom evolution more broadly. Sequencing the transcriptome of venom-producing glands has greatly increased the ability of researchers to examine and characterize the toxin repertoire of small taxa with low venom yields. Here, we use RNA-seq to characterize the Duvernoy's gland transcriptome of the Plains Black-headed Snake, Tantilla nigriceps, a small, semi-fossorial colubrid that feeds on a variety of potentially dangerous arthropods including centipedes and spiders. We generated transcriptomes of six individuals from three localities in order to both characterize the toxin expression of this species for the first time, and to look for initial evidence of venom variation in the species. Three toxin families-three-finger neurotoxins (3FTxs), cysteine-rich secretory proteins (CRISPs), and snake venom metalloproteinases (SVMPIIIs)-dominated the transcriptome of T. nigriceps; 3FTx themselves were the dominant toxin family in most individuals, accounting for as much as 86.4% of an individual's toxin expression. Variation in toxin expression between individuals was also noted, with two specimens exhibiting higher relative expression of c-type lectins than any other sample (8.7-11.9% compared to <1%), and another expressed CRISPs higher than any other toxin. This study provides the first Duvernoy's gland transcriptomes of any species of Tantilla, and one of the few transcriptomic studies of RFS not predicated on a single individual. This initial characterization demonstrates the need for further study of toxin expression variation in this species, as well as the need for further exploration of small RFS venoms.


Assuntos
Colubridae/metabolismo , Venenos de Serpentes/metabolismo , Toxinas Biológicas/metabolismo , Transcriptoma , Animais , Colubridae/genética , Metaloproteases/genética , Metaloproteases/metabolismo , Toxinas Biológicas/genética
6.
Acta Cir Bras ; 36(4): e360401, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34008743

RESUMO

PURPOSE: Quantify the tissue content of metalloproteinase-9 (MMP-9) and collagen in colic mucosa with and without intestinal transit after infliximab administration in rats subjected to Hartmann's surgery. METHODS: Twenty-two rats underwent colon diversion by Hartmann's surgery. Animals were maintained with intestinal bypass for 12 weeks to induce development of diversion colitis (DC). Afterwards, animals were divided into three groups: first group received subcutaneous application of saline solution (SS) 0.9%, while the remaining two groups received infliximab subcutaneously at doses of 5 or 10 mg·kg-1·week-1 for five consecutive weeks. After the intervention, animals were sacrificed, removing the segments with and without intestinal transit. Diversion colitis was diagnosed by histological study, and its intensity was determined by a validated inflammatory scale. Tissue expression of MMP-9 was assessed byimmunohistochemistry, while total collagen was assessed by histochemistry. Tissue content of both was measuredby computerized morphometry. RESULTS: Colon segments without intestinal transit had a higher degree of inflammation, which improved in animals treated with infliximab. Collagen content was always lower in those without intestinal transit. There was an increase in the collagen content in the colon without transit in animals treated with infliximab, primarily at a dose of 10 mg·kg-1·week-1. There was an increase in the content of MMP-9 in the colon without fecal transit, and a reduction was observed in animals treated with infliximab, regardless of the dose used. CONCLUSIONS: Application of infliximab reduces inflammation, increases the total collagen content and decreases the content of MMP-9 in the colon without intestinal transit.


Assuntos
Colo , Mucosa Intestinal , Animais , Colágeno , Colo/cirurgia , Infliximab , Metaloproteases , Ratos , Ratos Wistar
7.
Arch Oral Biol ; 127: 105148, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34022546

RESUMO

OBJECTIVE: The aim of this study was to synthesize and characterize an experimental primer containing cationic lipid nanoparticles (NPL-chitosan) and to evaluate its properties. DESIGN: The NPL-chitosan were synthesized by emulsion and sonication method. The experimental primers were applied in dentin surface of fifty human molars. The experimental groups were: 1) application of commercial primer; 2) Primer containing 2% of Chlorhexidine (CHX) 3); Primer with 2% NPL-chitosan 4); Primer with 0.6 % of NPL-chitosan 5); Primer with 0.4 % of NPL-chitosan. A composite resin plateau was used for the analysis, where sections were made for making the dentin beams. The effect of experimental primer with cationic nanoparticles in the inhibition of matrix metalloproteinase (MMP) activity was carrying out by in situ zymography. For the Resin-Dentin Adhesive Strength and in situ Zymography analysis, was used the One-way analysis of variance (ANOVA) with significance level of 95 %. RESULTS: Spherical NPL-chitosan presented size below 220 nm, polydispersity index of 0.179 and zeta potential positive and was stable over 75 days. These nanoparticles showed antibacterial activity agsainst S. mutans with MIC of the 0.4 % and MBC of 0.67 %. In the Microtensile Strength, no statistical difference was observed between the experimental groups (p = 0.9054). The in situ zymography assay showed that the group with 2% of NPL-chitosan presented higher inactivation activity of MMPs compared to the other groups (p < 0.05). CONCLUSION: The experimental primer containing NPL-chitosan has antimicrobial activity, does not alter the adhesive resistance and inactivates MMPs present in dentin.


Assuntos
Quitosana , Colagem Dentária , Nanopartículas , Resinas Compostas , Cimentos Dentários/farmacologia , Dentina , Adesivos Dentinários , Humanos , Teste de Materiais , Metaloproteases , Cimentos de Resina , Resistência à Tração
8.
FASEB J ; 35(6): e21538, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33956347

RESUMO

Two chaperones, Atp23p and Atp10p, were previously shown to regulate the assembly of yeast mitochondrial ATP synthase, and extra expression of ATP23 was found to partially rescue an atp10 deletion mutant, by an unknown mechanism. Here, we identified that the residues 112-115 (LRDK) of Atp23p were required for its function in assisting assembly of the synthase, and demonstrated both functions of Atp23p, processing subunit 6 precursor and assisting assembly of the synthase, were required for the partial rescue of atp10 deletion mutant. By chasing labeling with isotope 35 S-methionine, we found the stability of subunit 6 of the synthase increased in atp10 null strain upon overexpression of ATP23. Further co-immunoprecipitation (Co-IP) and blue native PAGE experiments showed that Atp23p and Atp10p were physically associated with each other in wild type. Moreover, we revealed the expression level of Atp23p increased in atp10 null mutant compared with the wild type. Furthermore, we found that, after 72 hours growth, atp10 null mutant showed leaky growth on respiratory substrates, presence of low level of subunit 6 and partial recovery of oligomycin sensitivity of mitochondrial ATPase activity. Further characterization revealed the expression of Atp23p increased after 24 hours growth in the mutant. These results indicated, in atp10 null mutant, ATP10 deficiency could be partially complemented with increased expression of Atp23p by stabilizing some subunit 6 of the synthase. Taken together, this study revealed the two chaperones Atp23p and Atp10p coordinated to regulate the assembly of mitochondrial ATP synthase, which advanced our understanding of mechanism of assembly of yeast mitochondrial ATP synthase.


Assuntos
Metaloproteases/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Metaloproteases/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência
9.
Methods Mol Biol ; 2302: 1-20, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877619

RESUMO

Rhomboid proteases are a ubiquitous superfamily of serine intramembrane peptidases that play a role in a wide variety of cellular processes. The mammalian mitochondrial rhomboid protease, Presenilin-Associated Rhomboid Like (PARL), is a critical regulator of mitochondrial homeostasis through the cleavage of its substrates, which have roles in mitochondrial quality control and apoptosis. However, neither structural nor functional information for this important protease is available, because the expression of eukaryotic membrane proteins to sufficient levels in an active form often represents a major bottleneck for in vitro studies. Here we present an optimized protocol for expression and purification of the human PARL protease using the eukaryotic expression host Pichia pastoris. The PARL gene construct was generated in tandem with green fluorescent protein (GFP), which allowed for the selection of high expressing clones and monitoring during the large-scale expression and purification steps. We discuss the production protocol with precise details for each step. The protocol yields 1 mg of pure PARL per liter of yeast culture.


Assuntos
Metaloproteases/isolamento & purificação , Proteínas Mitocondriais/isolamento & purificação , Saccharomycetales/crescimento & desenvolvimento , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Metaloproteases/genética , Proteínas Mitocondriais/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/genética , Transformação Genética
10.
Nat Rev Nephrol ; 17(8): 513-527, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33879883

RESUMO

Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs) belong to the metzincin family of zinc-containing multidomain molecules, and can act as soluble or membrane-bound proteases. These enzymes inactivate or activate other soluble or membrane-expressed mediator molecules, which enables them to control developmental processes, tissue remodelling, inflammatory responses and proliferative signalling pathways. The dysregulation of MMPs and ADAMs has long been recognized in acute kidney injury and in chronic kidney disease, and genetic targeting of selected MMPs and ADAMs in different mouse models of kidney disease showed that they can have detrimental and protective roles. In particular, MMP-2, MMP-7, MMP-9, ADAM10 and ADAM17 have been shown to have a mainly profibrotic effect and might therefore represent therapeutic targets. Each of these proteases has been associated with a different profibrotic pathway that involves tissue remodelling, Wnt-ß-catenin signalling, stem cell factor-c-kit signalling, IL-6 trans-signalling or epidermal growth factor receptor (EGFR) signalling. Broad-spectrum metalloproteinase inhibitors have been used to treat fibrotic kidney diseases experimentally but more targeted approaches have since been developed, including inhibitory antibodies, to avoid the toxic side effects initially observed with broad-spectrum inhibitors. These advances not only provide a solid foundation for additional preclinical studies but also encourage further translation into clinical research.


Assuntos
Rim/metabolismo , Redes e Vias Metabólicas , Metaloproteases/metabolismo , Proteínas ADAM/metabolismo , Animais , Humanos , Nefropatias/metabolismo , Metaloproteinases da Matriz/metabolismo
11.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810118

RESUMO

Experimental evidence for enzymatic mechanisms is often scarce, and in many cases inadvertently biased by the employed methods. Thus, apparently contradictory model mechanisms can result in decade long discussions about the correct interpretation of data and the true theory behind it. However, often such opposing views turn out to be special cases of a more comprehensive and superior concept. Molecular dynamics (MD) and the more advanced molecular mechanical and quantum mechanical approach (QM/MM) provide a relatively consistent framework to treat enzymatic mechanisms, in particular, the activity of proteolytic enzymes. In line with this, computational chemistry based on experimental structures came up with studies on all major protease classes in recent years; examples of aspartic, metallo-, cysteine, serine, and threonine protease mechanisms are well founded on corresponding standards. In addition, experimental evidence from enzyme kinetics, structural research, and various other methods supports the described calculated mechanisms. One step beyond is the application of this information to the design of new and powerful inhibitors of disease-related enzymes, such as the HIV protease. In this overview, a few examples demonstrate the high potential of the QM/MM approach for sophisticated pharmaceutical compound design and supporting functions in the analysis of biomolecular structures.


Assuntos
Simulação de Dinâmica Molecular , Peptídeo Hidrolases/química , Inibidores de Proteases/química , Teoria Quântica , Algoritmos , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Metaloproteases/metabolismo , Estrutura Molecular , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Conformação Proteica , Serina Proteases/química , Serina Proteases/metabolismo , Termodinâmica
12.
Int J Mol Sci ; 22(6)2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33804608

RESUMO

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that have been associated not only with various cellular processes, such as embryonic development and adult wound healing but also enhanced tumor survival, angiogenesis, and metastatic spread. Proteolytic cleavage of these single-pass transmembrane receptors has been suggested to regulate biological activities of their ligands during growth and development, yet little is known about the proteases responsible for this process. In this study, we monitored the release of membrane-anchored FGFRs 1, 2, 3, and 4 in cell-based assays. We demonstrate here that metalloprotease-dependent metalloprotease family, ADAM10 and ADAM17. Loss- and gain-of-function studies in murine embryonic fibroblasts showed that constitutive shedding as well as phorbol-ester-induced processing of FGFRs 1, 3, and 4 is mediated by ADAM17. In contrast, treatment with the calcium ionophore ionomycin stimulated ADAM10-mediated FGFR2 shedding. Cell migration assays with keratinocytes in the presence or absence of soluble FGFRs suggest that ectodomain shedding can modulate the function of ligand-induced FGFR signaling during cell movement. Our data identify ADAM10 and ADAM17 as differentially regulated FGFR membrane sheddases and may therefore provide new insight into the regulation of FGFR functions.


Assuntos
Metaloproteases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Animais , Linhagem Celular , Movimento Celular , Ativação Enzimática , Células Epiteliais/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Família Multigênica , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Proteína Quinase C/metabolismo , Proteólise , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/genética
13.
Int J Mol Sci ; 22(7)2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808504

RESUMO

Prostate cancer remains a leading cause of cancer-related morbidity in men. Potentially important regulators of prostate cancer progression are members of the metzincin superfamily of proteases, principally through their regulation of the extracellular matrix. It is therefore timely to review the role of the metzincin superfamily in prostate cancer and its progression to better understand their involvement in this disease. A systematic-like search strategy was conducted. Articles that investigated the roles of members of the metzincin superfamily and their key regulators in prostate cancer were included. The extracted articles were synthesized and data presented in tabular and narrative forms. Two hundred and five studies met the inclusion criteria. Of these, 138 investigated the role of the Matrix Metalloproteinase (MMP) subgroup, 34 the Membrane-Tethered Matrix Metalloproteinase (MT-MMP) subgroup, 22 the A Disintegrin and Metalloproteinase (ADAM) subgroup, 8 the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) subgroup and 53 the Tissue Inhibitor of Metalloproteinases (TIMP) family of regulators, noting that several studies investigated multiple family members. There was clear evidence that specific members of the metzincin superfamily are involved in prostate cancer progression, which can be either in a positive or negative manner. However, further understanding of their mechanisms of action and how they may be used as prognostic indicators or molecular targets is required.


Assuntos
Metaloproteases/metabolismo , Metaloproteases/fisiologia , Neoplasias da Próstata/metabolismo , Proteínas ADAM/metabolismo , Proteínas ADAMTS/metabolismo , Matriz Extracelular/fisiologia , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Próstata/patologia , Inibidores Teciduais de Metaloproteinases/metabolismo
14.
Toxins (Basel) ; 13(4)2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807363

RESUMO

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.


Assuntos
Anticorpos Neutralizantes/farmacologia , Antivenenos/farmacologia , Venenos de Crotalídeos/antagonistas & inibidores , Crotalus , Desintegrinas/antagonistas & inibidores , Metaloproteases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Mordeduras de Serpentes/tratamento farmacológico , Regulação Alostérica , Animais , Especificidade de Anticorpos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Reações Cruzadas , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/imunologia , Modelos Animais de Doenças , Desintegrinas/imunologia , Desintegrinas/metabolismo , Hemorragia/enzimologia , Hemorragia/etiologia , Hemorragia/prevenção & controle , Humanos , Metaloproteases/imunologia , Metaloproteases/metabolismo , Camundongos Endogâmicos BALB C , Agregação Plaquetária/efeitos dos fármacos , Mordeduras de Serpentes/sangue , Mordeduras de Serpentes/enzimologia , Mordeduras de Serpentes/imunologia
15.
Toxicon ; 197: 12-23, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33872676

RESUMO

Snakebite envenoming is a neglected tropical disease affecting millions of people every year, especially in vulnerable rural populations in the developing world. Viperid snakes cause envenomings characterized by a complex pathophysiology which includes local and systemic hemorrhage due to the action of snake venom metalloproteinases (SVMPs). The pathogenesis of SVMP-induced systemic hemorrhage has not been investigated in detail. This study explored the pulmonary hemorrhage induced in a murine model by a P-III SVMP from the venom of Crotalus simus. Histological analysis revealed extravasation in the lungs as early as 15 min after intravenous injection of the toxin, and hemorrhage increased at 360 min. Western blot analysis demonstrated the cleavage of basement membrane (BM) proteins in lung homogenates and in bronchoalveolar lavage fluid, implying an enzymatic disruption of this extracellular matrix structure at the capillary-alveolar barrier. Likewise, alveolar edema was observed, with an increment in protein concentration in the bronchoalveolar lavage fluid, and a neutrophil-rich inflammatory infiltrate was present in the parenchyma of the lungs as part of the inflammatory reaction. Pretreatment of mice with indomethacin, pentoxifylline and an anti-neutrophil antibody resulted in a significant decrease in pulmonary hemorrhage at 360 min. These findings suggest that this P-III SVMP induces acute lung injury through the direct action of this enzyme in the capillary-alveolar barrier integrity, as revealed by BM degradation, and as a consequence of the inflammatory reaction that develops in lung tissue. Our findings provide novel clues to understand the mechanism of action of hemorrhagic SVMPs in the lungs.


Assuntos
Venenos de Crotalídeos , Metaloproteases , Animais , Membrana Basal , Venenos de Crotalídeos/toxicidade , Hemorragia/induzido quimicamente , Inflamação , Metaloproteases/toxicidade , Camundongos , Venenos de Serpentes
16.
J Bacteriol ; 203(13): e0010021, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33927050

RESUMO

Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts, including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to those grown in control cultures but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum, indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here, we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to posttranslationally inhibit zinc metalloprotease activity and thereby affect the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment. IMPORTANCE The factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases, suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.


Assuntos
Fibrose Cística/microbiologia , Complexo Antígeno L1 Leucocitário/metabolismo , Serina Endopeptidases/metabolismo , Escarro/microbiologia , Zinco/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Pulmão , Metaloendopeptidases/metabolismo , Metaloproteases , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Infecções Estafilocócicas , Staphylococcus aureus , Virulência , Fatores de Virulência
17.
Biomolecules ; 11(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669093

RESUMO

Osteoarthritis (OA) is one of the most common and widespread diseases which is highly disabling for humans. This makes OA a chronic disease for which it is urgent to find new therapeutic strategies. The inflammatory state in OA contributes to its progression through multiple mechanisms involving the recruitment of phagocytes and leukocytes, inflammatory response, and reactive oxygen species (ROS) production. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is classifiable as a piperidine nitroxide, with excellent antioxidant effects, while its anti-inflammatory role is not yet clear. On this basis, we explored its promising biological properties in two in vitro model:, macrophage (J774) and chondrocyte (CC) cell lines. With this aim in mind, we induced inflammation in J774 and CC using lipopolysaccharide (LPS) and Interleukin1ß (IL-1ß), and after 24, 72 and 168 h of tempol treatment analyzed their effects on cytotoxicity and anti-inflammatory activity. Our data suggested that tempol treatment is able to reduce inflammation and nitrite production in LPS-induced J774 as well as reducing the production of proinflammatory mediators including cytokines, enzymes, and metalloproteases (MMPs) in IL-1ß-stimulated CC. Thus, since inflammation and oxidative stress have a crucial role in the pathogenesis and progression of OA, tempol could be considered as a new therapeutic approach for this pathology.


Assuntos
Óxidos N-Cíclicos/uso terapêutico , Inflamação/tratamento farmacológico , Osteoartrite/tratamento farmacológico , Animais , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interferon beta/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteases/metabolismo , Osteoartrite/induzido quimicamente , Osteoartrite/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Marcadores de Spin
18.
PLoS One ; 16(3): e0248975, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33735297

RESUMO

Nuclear factor-kappa B (NF-κB) plays a critical role in the host defense against microbial pathogens. Many pathogens modulate NF-κB signaling to establish infection in their host. Salmonella enterica serovar Typhimurium (S. Typhimurium) possesses two type III secretion systems (T3SS-1 and T3SS-2) and directly injects many effector proteins into host cells. It has been reported that some effectors block NF-κB signaling, but the molecular mechanism of the inactivation of NF-κB signaling in S. Typhimurium is poorly understood. Here, we identified seven type III effectors-GogA, GtgA, PipA, SseK1, SseK2, SseK3, and SteE-that inhibited NF-κB activation in HeLa cells stimulated with TNF-α. We also determined that only GogA and GtgA are involved in regulation of the activation of NF-κB in HeLa cells infected with S. Typhimurium. GogA, GtgA, and PipA are highly homologous to one another and have the consensus zinc metalloprotease HEXXH motif. Our experiments demonstrated that GogA, GtgA, and PipA each directly cleaved NF-κB p65, whereas GogA and GtgA, but not PipA, inhibited the NF-κB activation in HeLa cells infected with S. Typhimurium. Further, expressions of the gogA or gtgA gene were induced under the SPI-1-and SPI-2-inducing conditions, but expression of the pipA gene was induced only under the SPI-2-inducing condition. We also showed that PipA was secreted into RAW264.7 cells through T3SS-2. Finally, we indicated that PipA elicits bacterial dissemination in the systemic stage of infection of S. Typhimurium via a T3SS-1-independent mechanism. Collectively, our results suggest that PipA, GogA and GtgA contribute to S. Typhimurium pathogenesis in different ways.


Assuntos
Proteínas de Bactérias/metabolismo , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Animais , Células HeLa , Humanos , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos CBA , NF-kappa B/metabolismo , Células RAW 264.7 , Infecções por Salmonella/metabolismo , Zinco/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-33676005

RESUMO

We investigated the antiophidic properties of isohemigossypolone (ISO), a naphthoquinone isolated from the outer bark of the Pachira aquatic Aubl. The inhibition of phospholipase A2, coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by Bothrops pauloensis venom (Pb) was investigated. For this, we use samples resulting from the incubation of Pb with ISO in different concentrations (1:1, 1:5 and 1:10 w/w), we also evaluated the condition of treatment using ISO after 15 min of venom inoculation. The activities of phospholipase A2, coagulant, fibrinogenolytic, hemorrhagic and myotoxic induced by the B. pauloensis venom were significantly inhibited when the ISO was pre-incubated with the crude venom. For in vivo neutralization tests, the results were observed even when the ISO was applied after 15 min of inoculation of the venom or metalloprotease (BthMP). Also, to identify the inhibition mechanism, we performed in silico assays, across simulations of molecular coupling and molecular dynamics, it was possible to identify the modes of interaction between ISO and bothropic toxins BmooMPα-I, Jararacussin-I and BNSP-7. The present study shows that naphthoquinone isohemigossypolone isolated from the P. aquatica plant inhibited part of the local and systemic damage caused by venom proteins, demonstrating the pharmacological potential of this compound in neutralizing the harmful effects caused by snakebites.


Assuntos
Bombacaceae/química , Venenos de Crotalídeos/antagonistas & inibidores , Naftoquinonas , Extratos Vegetais , Mordeduras de Serpentes/tratamento farmacológico , Animais , Masculino , Metaloproteases/metabolismo , Camundongos , Naftoquinonas/química , Naftoquinonas/farmacologia , Fosfolipases A2/metabolismo , Casca de Planta/química , Extratos Vegetais/farmacologia
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